Noncompaction of the Ventricular Myocardium Is Associated with a De Novo Mutation in the β-Myosin Heavy Chain Gene

Noncompaction of the ventricular myocardium (NVM) is the morphological hallmark of a rare familial or sporadic unclassified heart disease of heterogeneous origin. NVM results presumably from a congenital developmental error and has been traced back to single point mutations in various genes. The objective of this study was to determine the underlying genetic defect in a large German family suffering from NVM. Twenty four family members were clinically assessed using advanced imaging techniques. For molecular characterization, a genome-wide linkage analysis was undertaken and the disease locus was mapped to chromosome 14ptel-14q12. Subsequently, two genes of the disease interval, MYH6 and MYH7 (encoding the α- and β-myosin heavy chain, respectively) were sequenced, leading to the identification of a previously unknown de novo missense mutation, c.842G>C, in the gene MYH7. The mutation affects a highly conserved amino acid in the myosin subfragment-1 (R281T). In silico simulations suggest that the mutation R281T prevents the formation of a salt bridge between residues R281 and D325, thereby destabilizing the myosin head. The mutation was exclusively present in morphologically affected family members. A few members of the family displayed NVM in combination with other heart defects, such as dislocation of the tricuspid valve (Ebstein's anomaly, EA) and atrial septal defect (ASD). A high degree of clinical variability was observed, ranging from the absence of symptoms in childhood to cardiac death in the third decade of life. The data presented in this report provide first evidence that a mutation in a sarcomeric protein can cause noncompaction of the ventricular myocardium

Molecular genetic investigation of autosomal dominant muscular dystrophy

This thesis contributes to the Human Genome Project by adding detail to the physical and genetic maps of the human genome, and by identifying a strong candidate gene for a form of distal myopathy. Genomic clones for the human skeletal muscle genes slow troponin (TNN/1), alpha actin (ACTA1), and (3-tropomyosin (TPM2) were isolated for use in the fluorescent in situ hybridisation localisation of these genes on the cytogenetic map of the human genome. The localisation of these genes made them potential candidates for inherited skeletal muscle diseases, including the muscular dystrophies investigated here. Microsatellite, VNTR and RFLP markers were used in a search for linkage to a novel form of distal myopathy segregating in a Western Australian family. The decadic logarithm of the likelihood ratio, or \u27lod score\u27 method, was used to determine linkage between markers and this distal myopathy gene. A 22.4 cM candidate region was identified at 14q11.2. This was the first localisation of a distal myopathy gene. The Human Genome Organisation Nomenclature Committee reserved MPD1, \u27myopathy, distal 1 \u27, for this form of distal myopathy, now known as Laing myopathy. The MPD1 candidate region was excluded as the disease gene location for two other forms of distal myopathy. Silburn myopathy in 1994, which established the genetic heterogeneity of distal myopathy, and Felice myopathy in 1996. The exclusion of the MPD1 and French-Canadian OPMD candidate regions as disease gene locations for a putative-OPMD segregating in a Western Australian family, proved that this disease gene did not lie at 14q11.2. Testing an MPD1 muscle-specific candidate gene for the Laing myopathy mutation, the myosin heavy polypeptide 7 gene (MYH7), identified seven base changes between the MPD1 proband sequence and the published MYH7 eDNA sequence. All of these base changes were found in eight unrelated, unaffected Western Australians, therefore none of them were the Laing myopathy mutation. Two further differences to the published MYH7 sequence segregated exclusively with the MPD1 proband. One of these, the MYH7 G5073C (cDNA)/G23628C (gDNA) base change, caused a critical change to the MYH7 13-myosin heavy chain polypeptide product (13-MyHC). An A 1663P 13-MyHC substitution. G23628/C 23628 segregated with Laing myopathy in the Western Australian distal myopathy family. This segregation was confirmed by a single-strand conformation polymorphism test, then used to test 256 unaffected chromosomes. None possessed MYH7C23628. Two patients from European distal myopathy families phenotypically similar to Laing myopathy, the Voit and Scoppetta families, were tested for the presence of MYH7 gDNA G23628/C23628 heterozygosity. Both were homozygous MYH7 G23628. One of these patients (Voit) was also tested for MYH7 eDNA G5073/C5073 heterozygosity. She was homozygous MYH7 G5073. An analysis of the effect of the 13-MyHC A 1663P substitution at various levels of protein structure strengthened the candidature of MYH7 G5073C as the Laing myopathy mutation. It demonstrated the extreme rarity of the 13-MyHC A 1663P substitution; it showed that this substitution did have a detrimental effect on coiled-coil formation; and it identified ways in which the 13-MyHC A 1663P substitution could disrupt myofibrillogenesis or contractility. Future research directions are identified and the contribution of this work to evolving concepts in muscular dystrophy is evaluated

A Combined Linkage and Exome Sequencing Analysis for Electrocardiogram Parameters in the Erasmus Rucphen Family Study

Electrocardiogram (ECG) measurements play a key role in the diagnosis and prediction of cardiac arrhythmias and sudden cardiac death. ECG parameters, such as the PR, QRS, and QT intervals, are known to be heritable and genome-wide association studies of these phenotypes have been successful in identifying common variants; however, a large proportion of the genetic variability of these traits remains to be elucidated. The aim of this study was to discover loci potentially harboring rare variants utilizing variance component linkage analysis in 1547 individuals from a large family-based study, the Erasmus Rucphen Family Study (ERF). Linked regions were further explored using exome sequencing. Five suggestive linkage peaks were identified: two for QT interval (1q24, LOD = 2.63; 2q34, LOD = 2.05), one for QRS interval (1p35, LOD = 2.52) and two for PR interval (9p22, LOD = 2.20; 14q11, LOD = 2.29). Fine-mapping using exome sequence data identified a C > G missense variant (c.713C > G, p.Ser238Cys) in the FCRL2 gene associated with QT (rs74608430; P = 2.8 x 10(-4), minor allele frequency = 0.019). Heritability analysis demonstrated that the SNP explained 2.42% of the trait's genetic variability in ERF (P = 0.02). Pathway analysis suggested that the gene is involved in cytosolic Ca2+ levels (P = 3.3 x 10(-3)) and AMPK stimulated fatty acid oxidation in muscle (P = 4.1 x 10(-3)). Look-ups in bioinformatics resources showed that expression of FCRL2 is associated with ARHGAP24 and SETBP1 expression. This finding was not replicated in the Rotterdam study. Combining the bioinformatics information with the association and linkage analyses, FCRL2 emerges as a strong candidate gene for QT interval

Doctor of Philosophy

dissertationEvery year millions of children are born with a birth defect. Birth defects, which can be described as abnormalities of structure or function that is present from birth, are the leading cause of infant death in developed countries and a significant cause of morbidity and economic burden in low- or middle-income countries. This dissertation addresses the genetic basis of distal arthrogryposes (DAs), a subgroup of birth defects that are characterized by contractures of the distal joints of a limb. Based on previous research of our laboratory, we hypothesized that DAs are defects of contractile apparatus in fast twitch skeletal myofibers and tested this hypothesis in four DA syndromes. We found that mutations of the embryonic myosin heavy chain gene cause DA2A and DA2B, whereas a missense mutation of the perinatal myosin heavy chain gene is responsible for DA7. Furthermore, we found mutations in the adult and extraocular myosin heavy chain genes in some DA5 patients. Furthermore, we noticed some patients with similar findings who do not meet the diagnostic criteria of the known DA syndromes. We proposed one of these conditions to be named as DA10, and mapped this condition to the long arm of chromosome 2. We named the other condition as the CATSHL syndrome, which we showed to be caused by a loss-of-function mutation in the fibroblast growth factor receptor 3 gene. The main contribution of this research is to benefit affected individuals and their families, since molecular testing can now be offered to them. In addition, through further studies leading to a better understanding of normal and abnormal development, effective strategies for prevention and treatment of congenital limb malformations can be developed

Thick and Thin Filament Gene Mutations in Striated Muscle Diseases

The sarcomere is the fundamental unit of cardiac and skeletal muscle contraction. During the last ten years, there has been growing awareness of the etiology of skeletal and cardiac muscle diseases originating in the sarcomere, an important evolving field. Many sarcomeric diseases affect newborn children, i. e. are congenital myopathies. The discovery and characterization of several myopathies caused by mutations in myosin heavy chain genes, coding for the major component of skeletal muscle thick filaments, has led to the introduction of a new entity in the field of neuromuscular disorders: myosin myopathies. Recently, mutations in genes coding for skeletal muscle thin filaments, associated with various clinical features, have been identified. These mutations evoke distinct structural changes within the sarcomeric thin filament. Current knowledge regarding contractile protein dysfunction as it relates to disease pathogenesis has failed to decipher the mechanistic links between mutations identified in sarcomeric proteins and skeletal myopathies, which will no doubt require an integrated physiological approach. The discovery of additional genes associated with myopathies and the elucidation of the molecular mechanisms of pathogenesis will lead to improved and more accurate diagnosis, including prenatally, and to enhanced potential for prognosis, genetic counseling and developing possible treatments for these diseases. The goal of this review is to present recent progress in the identification of gene mutations from each of the major structural components of the sarcomere, the thick and thin filaments, related to skeletal muscle disease. The genetics and clinical manifestations of these disorders will be discussed

Herculin, a Fourth Member of the MyoD Family of Myogenic Regulatory Genes

We have identified and cloned herculin, a fourth mouse muscle regulatory gene. Comparison of its DNA and deduced amino acid sequences with those of the three known myogenic genes (MyoD, myogenin, and Myf-5) reveals scattered short spans with similarity to one or more of these genes and a long span with strong similarity to all three. This long span includes a sequence motif that is also present in proteins of the myc, achaete-scute, and immunoglobulin enhancer-binding families. The herculin gene is physically linked to the Myf-5 gene on the chromosome; only 8.5 kilobases separate their translational start sites. A putative 27-kDa protein is encoded by three exons contained within a 1.7-kilobase fragment of the herculin gene. When expressed under the control of the simian virus 40 early promoter, transfected herculin renders murine NIH 3T3 and C3H/10T1/2 fibroblasts myogenic. In doing so, it also activates expression of myogenin, MyoD, and endogenous herculin in NIH 3T3 recipients. In adult mice, herculin is expressed in skeletal muscle but is absent from smooth muscle, cardiac muscle, and all nonmuscle tissues assayed. Direct comparison of the four known myogenic regulators in adult muscle showed that herculin is expressed at a significantly higher level than is any of the others. This quantitative dominance suggests an important role in the establishment or maintenance of adult skeletal muscle

A combined linkage and exome sequencing analysis for electrocardiogram parameters in the Erasmus Rucphen family study

Electrocardiogram (ECG) measurements play a key role in the diagnosis and prediction of cardiac arrhythmias and sudden cardiac death. ECG parameters, such as the PR, QRS, and QT intervals, are known to be heritable and genome-wide association studies of these phenotypes have been successful in identifying common variants; however, a large proportion of the genetic variability of these traits remains to be elucidated. The aim of this study was to discover loci potentially harboring rare variants utilizing variance component linkage analysis in 1547 individuals from a large family-based study, the Erasmus Rucphen Family Study (ERF). Linked regions were further explored using exome sequencing. Five suggestive linkage peaks were identified: two for QT interval (1q24, LOD = 2.63; 2q34, LOD = 2.05), one for QRS interval (1p35, LOD = 2.52) and two for PR interval (9p22, LOD = 2.20; 14q11, LOD = 2.29). Fine-mapping using exome sequence data identified a C > G missense variant (c.713C > G, p.Ser238Cys) in the FCRL2 gene associated with QT (rs74608430; P = 2.8 × 10-4, minor allele frequency = 0.019). Heritability analysis demonstrated that the SNP explained 2.42% of the trait's genetic variability in ERF (P = 0.02). Pathway analysis suggested that the gene is involved in cytosolic Ca2+ levels (P = 3.3 × 10-3) and AMPK stimulated fatty acid oxidat